Team:Hong Kong HKUST/TimeControl
Team Hong Kong - HKUST
Time Control Module
Overview / Rationale
The purpose of the Time Control module is to introduce a significant delay between the induction of the Sensing module and the activation of the Recombination module, which allows time for AHL to sufficiently amplify and diffuse to other cells by quorum sensing, as well as allocates time for gene-of-interest expression. The control of the delay is executed by employing transcriptional and translational delay of cI and phlF repressors.
Time module has significant application in genetic containment strategy. The amount of protein-of-interest can be controlled by the delay in Cre recombinase expression, which will knock out the construct and terminate the protein-of-interest production process. This provides a secure and safe method for researchers to control the expression within genetic modified organism (GMO).
Diagram 1. Expression in time module construct in the absence of induction by AHL molecules
Diagram 2. Expression in time module construct with induction by AHL moleculesOur Design
Our concept comes from a two-step cascade study which used IPTG as an inducer that regulates the system involving LacI repressor and Tet repressor with their corresponding repressible promoters. The experiment shows a delay of about one cell-cycle per step (ref 1). Our simulated graph shown in modelling (4.2 : Adding Time Control) is also based on the concept of two-step cascade study. In our design, we control Cre recombinase to express upon AHL induction.
The construct starts with pLuxR which is inducible when AHL-LuxR dimer binds. The major CDS in the Time Module construct just downstream pLuxR (BBa_R0062) are lambda cI repressor (BBa_C0051) and PhlF repressor (BBa_K1725040). Both are able to repress their downstream constitutive promoters separated by double terminators. These promoters are pcI (BBa_R0051) and pPhlF (BBa_K1725000) respectively. The two pairs of repressors and promoters do not work in synchrony i.e. when cI expresses, PhlF is repressed and vice versa.
Since the purpose of this time delay is to turn Cre recombinase either on or off dependent on the presence of AHL, there are two scenarios of activity in Time Module Construct:
- In the absence of AHL, only PhlF molecules produce and repress its pPhlF. Then, the expression of downstream recombinase is rendered.
- The second scenario is when sufficient AHL initiates cI protein production. The cI molecules will inhibit PhlF expression by repressing pcI. This turns on pPhlF, leading to expression of recombinase.
In order to observe the maximum and minimum time for the delay in Cre recombinase expression, ribosomal binding site (RBS) downstream to pLuxR is adjusted. In iGEM biobricks, multiple strengths of RBS can be sought. We selected five different ribosomal strengths, ranging from the strongest to the weakest:
The stronger the RBS strength, the higher the recombinase expression. We expect that transcriptional and translational delay of repressor coupled with tuning ribosomal strength to the lowest degree will significantly contribute to the longest delay of Cre expression.
Planned characterization
LuxR cassettes expresses LuxR protein that can form dimers with AHL. This dimer is the inducer for pLuxR. In order to see the effect of different RBS strength used in our time control construct, we use YFP whose expression can be quantified by fluorescence level.
